N-Maleoyl amino acids and N-maleoyl peptides, as N-alkylmaleimides, are able to react rapidly and specifically with thiol groups. Carboxyl activated esters of N-maleoyl amino acids, such as, for example, the N-hydroxysuocinimide esters, are able to react rapidly with amino groups. The activated esters of N-(maleoyl amino acids, therefore, are particularly useful for linking compounds containing amino groups on the one hand to compounds containing thiol groups on the other hand. The utility of such compounds in preparing peptide-protein conjugates and a wide variety of other conjugates of biochemical interest in this manner have been disclosed in the following references: Keller et al., Helv. Chim. Aota, 58, 531 (1975); Wuensch et al., Biol. Chem. Hoppe-Seyler, 366, 53 (1985): Moroder et al., Biopolymers, 22, 481 (1983); Yoshitake et al., Eur. J. Biochem., 101, 395 (1979); Fujiwara et al., Cancer Research, 41, 4121 (1981); and Rich et al., J. Med. Chem., 18, 1004 (1975).
These same references also discuss the preparation of N-maleoyl amino acids and their esters. Keller et al., in one of the earliest of the above publications, reported that although maleamio acids are readily accessible by the reaction of amino acids with maleic anhydride, their subsequent cyclization to the maleimide is difficult in the presence of an additional free carboxyl group. Although maleamic acid esters had been successfully cyclized to the maleimides, Rich et al., in another of the earliest of the above publications, noted that the preferential hydrolysis of the maleimide effectively prevented the preparation of N-maleoyl amino acids from their esters. More recent approaches have appeared in the literature that avoid these difficulties in preparing activated esters of N-maleoyl amino acids. Most of these approaches require either multiple reaction steps and/or the use of relatively expensive dicyclohexyl carbodiimide. The present invention provides a convenient means of rapidly preparing the activated esters of certain N-maleoyl amino acids directly without the use of dicyclohexyl carbodiimide.